PROJECT SUMMARY Aging leads to progressive alterations in extracellular matrix architecture and composition that increases elastic modulus, contributing prominently to declines in tissue and organ function, and to progressive fibrosis in various disease contexts. Identifying mechanisms by which to interrupt, and potentially reverse, aging related matrix stiffness changes represents an important goal. Recent studies have identified pivotal roles for Yes- associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) transcription co-factors in activating fibroblasts to drive matrix deposition and remodeling in the lung, liver, kidney, muscle and skin. YAP and TAZ are both responsive to their matrix mechanical environments, and critical effectors that promote cell-mediated changes to matrix stiffness, highlighting their potential role as targets in aging-related stiffness changes. However, YAP and TAZ are widely expressed and important in a wide array of cell functions, including stem cell maintenance, and epithelial and endothelial homeostasis. Thus, efforts to alter matrix stiffening require selective targeting YAP and TAZ in fibroblasts. The goal of this High Throughput Screening (HTS) proposal is to discover novel chemical probes that allow selective targeting of YAP/TAZ in human fibroblasts, following the approach outlined in these three specific aims: (1) Miniaturize and perform high- throughput screen to identify fibroblast-selective inhibitors of YAP and TAZ using high-content imaging of native YAP/TAZ nuclear localization in human fibroblasts; (2) Validate the biological activity of fibroblast- selective hits in secondary and functional assays; and (3) Profile selected compounds using in vitro and in vivo assays and Mechanism-of-Action (MOA) studies. Compounds representing validated and fibroblast-selective scaffolds of interest will be profiled in vitro using primary human lung, kidney, hepatic and cardiovascular mesenchymal cells to ensure activity in primary fibroblasts from organs that experience age-dependent matrix stiffening, and in fibroblasts from patients with IPF, a fatal interstitial lung disease closely linked to aging. Up to three top validated hits will be tested for proof of therapeutic concept in an in vivo assay using the bleomycin animal model of pulmonary fibrosis. The proposed work has a high potential of identifying fibroblast-selective compounds inhibiting YAP-TAZ activation and could lead to novel therapeutics for IPF and other fibrotic diseases as well as molecular probes to further investigate aging-related stiffness changes of the ECM.